Magnetic particles with a closed ultrathin silica layer, method for the production thereof and their use

ABSTRACT

The invention relates to magnetic particles coated with silica (Si02), whereby the silicate layer is closed and tight and is characterized by having an extremely small thickness on the scale of a few nanometers—hereafter also referred to as a silica nanolayer. This invention also relates to an improved method for producing these silicate-containing magnetic particles that, in comparison to the prior art, lead to a product having a closed silicate layer and thus entail a highly improved purity. In addition, the novel method prevents an uncontrolled formation of aggregates and clusters of silicates on the magnetite surface, thereby having a positive influence on the properties and biological applications cited below. The novel method also enables the depletion of nanoparticulate solid substance particles on the basis of a fractionated centrifugation. The inventive magnetic particles exhibit an optimized magnetization and suspension behavior as well as a very advantageous run-off behavior from plastic surfaces. These highly pure magnetic particles coated with silicon dioxide are preferably used for isolating nucleic acids from cell and tissue samples, whereby the separating out from a sample matrix ensues by means of magnetic fields. These particles are particularly well suited for the automatic purification of nucleic acids, mostly from biological body samples for the purpose of analyzing them with different amplification methods.

In recent times, molecular diagnostics have become increasinglyimportant. Molecular diagnostics have entered into the clinicaldiagnosis of illnesses. This includes the measurement of molecularmarkers to improve the diagnosis of a disease, early detection, themonitoring of an illness during therapy, the prognosis of illnesses andthe prediction of effects or side-effects of medicines (including thedetection of infective agents, detection of mutations of the genome, theprediction of effects and side-effects of medicines on the basis ofpredetermined genetic patterns or those acquired in the course of anillness, detection of circulating tumor cells and the identification ofrisk factors for predisposition to an illness). Methods of moleculardiagnosis have meanwhile also been used in veterinary medicine, analysisof the environment and foodstuff testing. A further application area isinvestigations by pathological/cytological institutes or in the courseof forensic investigations. Genetic diagnosis has meanwhile also beenused as part of healthcare (e.g. investigation of banked blood forfreedom from infective agents), and legislation is planned to regulatesuch tests. Methods which are also used in clinical molecular diagnosis(such as hybridization or amplification techniques such as PCR(polymerase chain reaction), TMA (transcription mediated amplification),LCR (ligase chain reaction), bDNA (branched DNA) or NASBA (nucleic acidsequence based amplification) also form part of routine procedures inbasic scientific work.

A precondition for performing an assay in molecular diagnostics isgenerally the isolation of DNA or RNA from the sample to be analyzed.There are of course analysis methods, such as bDNA-based tests thatenable nucleic acid isolation and detection reaction to be carried outat the same time but PCR, as the most widely used molecular biologicalmethod in molecular diagnostics, almost always requires the use ofpreviously purified nucleic acids because of their capacity to beinfluenced by exogenic factors.

The conventional preparation process for nucleic acids is in this casebased on a fluid-fluid extraction. An example of this is thephenol-chloroform extraction of DNA from body samples. However, thegreat effort required and the need to sometimes perhaps use highly toxicsubstances means that this method has fallen considerably into disfavorin recent years compared with solid-phase based methods.

With the use of solid-phase based extraction methods for nucleic acids,the sample preparation can be subdivided into the actual analysisoperation, largely independent of the particular problem, into fourbasic steps: 1. Conditioning of the solid phase; 2. Selective orspecific bonding of the analytes to the solid phase and removal of theremaining sample matrix; 3. Washing out any impurities from the solidphase and 4. Elution of the enriched and purified analytes.

The well known property of nucleic acids to bond specifically tosilicate-containing adsorbents such as glass powder [Proc. Natl. Acad.USA 76 (1979) 615-619, Anal. Biochem. 121 (1982) 382-387], diatomaceousearth [Methods Enzymol. 65 (1979) 176-182] or native silicon dioxide [J.Clin. Microbiol. 28 (1990) 495-503, EP 0 389 063 B1] under chaotropic orhigh-salt conditions, i.e. at high concentrations of chaotropes or othersalts, has long been used for the selective and reversible bonding ofnucleic acids. With the aid of a buffer containing water-soluble organicsolvent, usually a low aliphatic alcohol, impurities are then washedfrom the adsorbent, the carrier is dried and the adsorbed nucleic acidsare eluated with distilled water or a so-called low-salt buffer, i.e. abuffer with a low ion strength.

In view of the complete and cost-effective automation of nucleic acidisolation, methods with super-paramagnetic adsorbents play anincreasingly important role.

In the simplest example (WO 01/46404), commercially produced magneticparticles produced for technical applications, such as electrographictoners, are used directly for nucleic acid preparation without furthermodification.

Products of this kind produced by technical mass production do, however,meet one of the most important preconditions, such as a specific nucleicacid absorption and magnetisability. On the other hand, thesecommercially available products are unable to meet important boundaryconditions that are indispensable for highly-sensitive and reproducibleresults. For example, it is of decisive importance in the field of virusdiagnostics (e.g. HCV or HIV) to extract the viral nucleic acidsquantitively from the serum or plasma, i.e. with almost 100% yield, inorder from this to derive an accurate virus concentration in theserum/plasma and thus make decisions with regard to therapy. The purityof the magnetic particles also plays a decisive role with regard tooptical evaluation. Especially with magnetite particles that arefrequently still micro-porous, diffusion of iron atoms from theparticles can lead to colored solutions that can severely disturb thetransmission or reflection measurements.

For this reason, various developments of magnetic particles forbiological applications, particularly with regard to the manual andautomated isolation of nucleic acid, are described.

In this case, magnetic particles that support a high density of SiOHgroups on the surface play an outstanding role. It is known that SiOHgroups can form reversible bonds with nucleic acids. Silica-modifiedmagnetic particles are also the object of this invention.

To obtain highly-sensitive, quantitative and reproducible results, suchmagnetic particles must, in addition to magnetizability and the capacityto bond with nucleic acid, fulfill further boundary conditions, whichare described in more detail in the following.

Particle Size and Particle Size Distribution

It has been shown that magnetic particles of Fe₃0₄ (magnetite), forelectrographic toner with primary particle sizes of approximately 0.1 to1 μm, e.g. available from the Lanxess company under the name Bayoxide E,meet almost ideal preconditions with regard to particle size. Suchparticle sizes enable the important boundary condition of “suspensionstability” important for biological applications to be achieved. Thismust on the one hand be sufficiently resistant to ensure that nosignificant sedimentation occurs within a few minutes, for example tento fifteen minutes (adsorption of nucleic acids) after shaking, whereasthe magnetic particles loaded with nucleic acids must be able to becompletely separated as regards the shortest possible analysis timeswithin a few minutes, for example within one to five minutes.

However, the available magnetic particles of Fe₃0₄ (magnetite)unfortunately still have ongoing deficiencies in this respect, in thatsmall amounts of very fine magnetite particles in the nanometer rangeare still present.

These unwanted by-products that because of their large surface can bondconsiderable amounts of nucleic acids are unfortunately not separated inthe magnetic field within a few minutes and thus the information contentof these nucleic acids, especially with regard to the quantitivemeasurement of nucleic acids, can be lost.

In addition to these losses in yield, this also often leads to unclear,often yellow-brown, supernatants that can not only negatively influencethe commercial marketing but can also interfere with the photometricevaluation of the eluates.

It would therefore be of great advantage if this “nanoparticle magnetiteparticulate component” could be separated for the biological applicationdescribed here.

Silicate Content:

As mentioned above, some magnetic particles produced on a largetechnical scale, for example the Bayoxide E series from the Lanxesscompany, still have a certain nucleic acid bonding capacity even withoutspecial silica post-treatment, because they are produced in bulk andtherefore also support SiOH groups on the surface in small amounts.Because of the low nucleic acid adsorption capacity, such productsrequire corresponding relatively large amounts of magnetic particles,which means that the preparation of small sample volumes is hampered.

Furthermore, such products have a wetting behavior of vessel walls, suchas glass or plastic walls of microtiter plates such as are routinelyused for nucleic acid purification, that is unfavorable for theapplication described here. Therefore substantial amounts of theunmodified, relatively hydrophobic magnetic particles remain adsorbed inaqueous suspensions on the microtiter plate walls and thus lead toinaccuracies in pipetting and loss of yield.

Particles with a high density of SiOH surface groups, which because oftheir hydrophilicity very advantageously roll off plastic walls inparticular, such as the aforementioned microtiter plates, behave veryfavorably in this respect.

With many magnetic particle developments for the isolation of nucleicacid, the silica proportion is accordingly dominant compared with themagnetite proportion. As, for example, described in WO 01/71732, silicaparticles that can be magnetized by the magnetite inclusion are obtainedby hydrolysis from reactive silica compounds such as tetraethoxysilane(TEOS) in the presence of magnetite particles. Because of the highdensity of SiOH groups on the surface, such particles however show ahigh nucleic acid bonding capacity and a favorable wetting behavior ofthe microtiter plate walls, but on the other hand the magneticproperties are very heavily reduced corresponding to the reducedmagnetite content. Furthermore, the magnetic silica particles producedin this way have significantly more unfavorable morphologicalproperties, such as very heterogeneous particle sizes and particle sizedistribution and it should be mentioned that large non-sphericalparticles can lead to blockages during automatic pipetting.

Extractable Components:

The nucleic acids isolated using the magnetic particle process aregenerally subject to further processes such as a PCR (polymerase chainreaction), TMA (transcription mediated amplification), LCR (ligase chainreaction) or NASBA (nucleic acid sequence based amplification). Theseare highly-sensitive, enzyme-controlled processes that can be disturbedby numerous impurities and iron compounds, that, for example, can act asenzyme toxins.

Therefore, the magnetic particles produced for the nucleic acidpurification must fulfill particular purity requirements. If ironoxides, such as Bayoxide from the Lanxess company produced usingtechnical mass production, are used this problem is certainly notinsignificant because the magnetite particles have a certain porosityand surface roughness. Therefore, impurities can become included in themicropores both from the process of iron oxide production and in thesucceeding silica treatment that as enzyme toxins or in the case ofcolored impurities can interfere with the photometric evaluation duringsubsequent processes.

Object of this Invention

The object of this invention is to produce, on the basis of commerciallyavailable magnetic particles, silica-modified magnetic particles with ahigh density of SiOH surface groups and a closed and tight surface layerof silicate. Neither the morphology nor the very good magneticproperties of the initial products should be substantially influenced bythe silica modification. Equally, the wetting behavior on plasticsurfaces should be positively influenced by the silica coating.Furthermore, the silica-modified magnetic particles should be optimizedwith regard to extractable impurities to the extent that the release ofimpurities or iron compounds from the magnetite core is prevented and nointerference is possible with either the biological detection reactionsor the photometric evaluation.

Nearest Prior Art

On the basis of Bayoxide E magnetic particles from the Lanxess company,WO 03/058649 describes a smart process for silica deposition on theparticle surface using sodium silicate solutions, for example sodiumsilicate HK 30 from the Cognis company. By a gradual dilution of the pHvalue in the Bayoxide E/sodium silicate, equivalent to a gradual pHshift from strong alkaline (pH11.5) to neutral (pH7), a carefuldeposition of silica on the magnetic particle surface takes place. If,as mentioned in WO 03/058649, the pH reduction takes place due to theaddition of acids (WO 98/31840), uncontrolled conversion of sodiumsilicate into silica (SiO₂) can occur at the acid infusion point withmagnetic particles becoming stored in the structure of the silica, sothat the aforementioned controlled silica deposition on the magneticparticle surface is by no means achieved. Nonetheless, the formation ofminute silica aggregates or clusters on the surface cannot be completelyprevented by the “batch method” described in WO 03/058649.

Whereas the silica-modified magnetic particles described in WO 03/058649have good properties with regard to surface structure and nucleic acidbonding behavior, very disadvantageous yellow-brown supernatants can beobserved in the long-term behavior (after standing for a few weeks) ofthe relevant aqueous suspensions. In biological assays during whichsurfactants are generally used, this effect can be observed even aftershort stand times. An analysis shows that, in addition to sodiumsilicate components, traces of iron compounds and very fine magnetiteparticles can be found in these colored supernatants. Clearly, theseimpurities became locked into the porous magnetic particle structurethrough the silica surface, from where they diffuse outwards in thecourse of time. These observations also indicate that the silicate layeris not completely closed or is irregularly distributed by the batchmethod described in WO 03/058649 and therefore cannot prevent therelease of iron compounds.

DETAILED DESCRIPTION OF THIS INVENTION

In view of the elimination of the aforementioned extractable impurities,the technical process described in the following was optimized, with theprogress compared with the method described in WO 03/058649 beingdocumented. However, with the tests described here, in contrast to theexamples of WO 03/058649, Bayoxide E 8707, which is no longer availableas a standard product, was replaced by the very similar Bayoxide E 8706type. In both cases it is Fe₃0₄ magnetite that has a low Si content dueto its production, with type 8707 having an Fe/Si content of 99.1/0.9and Bayoxide E 8706 having 99.4/0.4. The surface quality, particularlythe pH value of the Fe₃0₄ magnetite, is important for the methodaccording to the invention. Whereas Bayoxide E 8707 with a pH of 6.5 hasa slightly acid surface, a neutral pH value, or depending on the batcheven a slightly alkali value (pH 7.5), is found with the Bayoxide E 8706now used. Surprisingly, it was found that even these slightly alkalisurface properties can induce sodium silicate deposition. Normally, thesilica deposition takes place from the very alkali sodium silicatesolutions by the addition of acids.

Comparison tests then surprisingly showed that distinctly better resultscould be obtained with regard to extractable components if instead ofthe gradual pH reduction described in WO 03/058649 a continuous method,such as a membrane method was used. In this case, as described in moredetail in the examples, the aqueous sodium silicate/magnetic particlesuspension was purified after a reaction time of one hour using “crossflow microfiltration”. Cross flow microfiltration, which is carried outat a slight negative pressure, is, as described in “Basic Principles ofMembrane Technology” by M. Mulder, a known separation or purificationmethod. In this case the work is carried out at constant volumes, i.e.the permeate volume flow containing the impurities is replaced by thesame volume flow of incoming fresh water. In contrast to the dialysismethod known in biology, depending on the pore diameter not onlylow-molecular salts but also particulate impurities are separated duringmicrofiltration. This continuous cleaning process was continued untilthe quality of the outflowing permeate quality corresponded to thedegree of purity of the incoming fresh water, which took approximately12 to 15 hours depending on the size of the preparation.

During the analytical surface characterization using ESCA it was verysurprising to find that the silica-modified magnetic particles producedin this way have a novel, i.e. ultrathin, silica structure on the silicasurface, with which the improved purification or increased purity can becorrelated. This silica nanolayer is characterized by a silica layer ofup to 5 nm distributed uniformly over the complete particle surface.Furthermore, the method according to the invention however alsodescribes a layer thickness of 2 nm and also, quite particularlypreferred, layer thicknesses of 0.5 nm to 0.2 nm. The particles coatedin this way have a surface coating which is characterized in that it,for example, prevents the escape of irons into the surrounding solution.

The production of magnetic particles with a silica layer thickness of0.2 nm is described in example 3.

Furthermore, the inventive method is characterized by a closed and tightsilica layer, which is also associated with the improved purity orreduced observed contamination effect in the supernatant. The purity ofthese silica-coated magnetic particles produced according to theinventive method is substantially better compared with the methoddescribed in WO 03/058649. Thus, visible discoloration of thesupernatant after production and washing no longer occurs (see examples2 and 3). In particular, the tight and closed silica layer prevents theescape of visible, or also invisible, impurities, for example iron ions,which can disturb the amplification methods or the optical evaluation ofbiological experiments (see examples 4 and 5).

Furthermore, it was surprising to find that the formation of aggregatesand clusters of silicates on the magnetite surface was almost completelyprevented due to the slow and continuous dilution and thus reduction ofthe pH value to neutral values in the described membrane filtrationprocess and/or again strongly reduced compared to the “batch method”described in WO 03/058649. This well defined nanolayer of siliconpositively influences the properties and biological applicationsdescribed in the following.

Furthermore, it was also found that additional product optimization withrespect to clear supernatants could be achieved by carrying out afractionated centrifugation, which enabled a separation of slowlysedimenting iron oxide particles, after the membrane process.

With the samples produced in this way, which are treated as aqueoussuspensions, all criteria such as a magnetisability absolutely identicalto the initial product, unchanged morphology, high nucleic acid bondingcapacity, favorable roll-off from the walls of the microtiter plates andoutstanding stability of the suspension with trouble-free separation ofthe magnetic particles in the magnetic field within a few minuteswithout significant impurities in the supernatant are achieved.

The expression “magnetic particles coated with silica” includesmagnetite cores that are coated with a nanolayer of silica.

The expression “closed and tight silica layer” includes a uniform,homogenous single to multiple molecular silica layer in a range of lessthan 5 nm, with a layer thickness of 2 nm being particularly preferredand a layer thickness of 0.5 to 0.2 nm being quite particularlypreferred. This closed silica layer particularly prevents the release ofiron compounds and iron ions to the environment of the silica-coatedmagnetic particle.

The expression “improved methods of production” includes a washingprocess with the aid of a micro- or ultra-filtration unit that is easyto perform but is very intensive and leads to extreme purity of thesilica-coated magnetic particle. With this method, a slow, controlledand continuous dilution, and therefore a reduction of the pH value toneutral pH values in the reaction solution, occurs after an initialprecipitation of the nanolayer of silicate onto the particle surface,thus forming an extremely uniform, tight, closed and homogenous layer ofsilicate on the surface of the magnetite. Furthermore, unwantedformations of aggregates or clusters of silicates are prevented orlargely reduced.

The expression “depletion of nano particulate components with the aid ofthe centrifugation technique” includes the application of centrifugationtechniques or simple gravitational techniques. This producessedimentation of the required fractions, with it being possible toreject the unwanted nano particulate components by removing thesupernatant. By determining the particle size distribution usingultra-centrifugation, this effect can be detected by means of thedepleted minute fractions. With the centrifugation technique, theinitial suspension is centrifuged for fifteen minutes at approximately3000 g, the supernatant is removed and an equal amount of water orbuffer is added and then re-suspended and this step is repeated severaltimes (up to ten times). The gravitation technique simply means thatinstead of the centrifugation a long time is allowed to elapse until alarge proportion of the particles has settled on the bottom of thevessel and the aqueous supernatant is then replaced.

The expression “optimum magnetization behavior” includes the property ofthe inventive particles to have the largest possible amount of magnetiteand thus be completely separated from the sample matrix during thepurification within a few minutes, for example within one to fiveminutes, when a magnetic field is applied from outside to a reactionvessel. This is particularly noteworthy with respect to the shortestpossible purification times in an automated process using a pipettingrobot and for the use of the cheapest possible magnets with a limitedmagnetic field strength as hardware components.

The expression “suspension behavior” includes the property of theinventive particles to behave in such a way that due to an optimum grainsize distribution no significant sedimentation occurs within a fewminutes, for example ten to fifteen minutes (adsorption phase of thenucleic acids) after shaking during the purification phase.

The expression “optimum run-off behavior from plastic surfaces” includesthe property the inventive particles have of a low affinity to theplastic articles used in biological purification processes due to ahydrophilic surface quality. The plastic articles used mainly includepolystyrene, polyethylene and polypropylene vessels or “microtiter”plates of comparable plastics of any shape or size. The specific silicalayer of the inventive magnetic particles enables a repellinginteraction with these plastic surfaces, so that the coated magneticparticles roll off these surfaces and undergo no great interactions,which in the end could lead to a loss of yield during a biologicalpurification process of nucleic acids.

The expression “isolation” means the purification of nucleic acids froma biological sample using the aforementioned silica-coated magneticparticles and is divided into the following steps.

-   -   a) Dissolving the sample in a reaction vessel with a lysis        buffer and, after incubation, adding a bonding buffer, which        preferably contains chaotropic salts, with        guanidin(ium)isothiocyanate being particularly preferred, of        high molarity    -   b) Adding silicate-coated magnetic particles    -   c) Incubating at a temperature at which the nucleic acid bonds        to the magnetic particles    -   d) Removing constituents that are not bonded from the reaction        preparation by applying a magnetic field, which separates the        magnetic particles from the surrounding fluid    -   e) Applying a washing buffer several times followed by the        removal of said buffer with magnetization of the particles for        cleaning unspecifically bonded molecules from the nucleic acid    -   f) Adding an elution buffer under conditions in which the        nucleic acid is separated from the magnetic particles    -   g) Separating the eluate with the nucleic acid after        re-application of a magnetic field.

The expression “automated purification” includes variations of theseprocesses in which the manual labour by humans is replaced eithercompletely or only partially in steps, especially with the biologicalbody sample being dissolved with a special buffer during the steps, theaddition of magnetic particles, the incubation at a specifictemperature, the removal of non-absorbed sample constituents, thewashing steps, the elution of bonded nucleic acids from the particles ata specific temperature and the separation of the eluate from theparticle suspension.

The expression “nucleic acids” includes oligomer and polymerribonucleotides or 2′-desoxy-ribonucleotides with a chain length of morethan 10 monomer units. The monomer units in nucleic acids are linked byphosphoric acid diester compounds between 3′- and 5′-hydroxyl groups ofadjacent monomer units and the 1′-atom of the respective carbohydratecomponent is glycosidically bonded to a heterocyclic base. Nucleic acidscan form double and triple strands due to the development ofintermolecular hydrogen bridge bonds.

This also includes protein/nucleic acid complexes and nucleic acids withsynthetic nucleotides such as morpholinos or PNAs (peptide-nucleicacids).

The expression “biological body sample” includes biological materialcontaining nucleic acid, such as whole blood, blood serum or bloodplasma, especially serum or plasma containing a virus, very particularlyserum samples infected with HIV and HCV, “Buffy Coat” (white blood cellfraction of the blood), faeces, ascites, swabs, sputum, organ aspirates,biopsies, tissue sections, in this case very particularly differentlyfixed tissue sections, especially those fixed with fixing agentscontaining formalin, and paraffin-embedded tissue sections, secretions,liquor, bile, lymphatic fluid, urine, stool, sperm, cells and cellcultures. This can also include nucleic acids that originate frombiochemical processes and are then to be purified.

The expression “detection with various amplification methods” includesthe duplication of purified nucleic acids using variousmolecular-biological technologies, especially PCR,transcription-mediated amplification (TMA), LCA or also NASBA and thesucceeding or simultaneous detection of the amplification products. Thisalso includes detection using signal amplification methods such as ofbDNA, i.e. without nucleic acid amplification. Detection of the PCR inparticular can be carried out by the application of kinetic methods withthe aid of fluorescence technology under real-time conditions or can becarried out using a conventional agarose gel. The real-time PCR inparticular enables a very good quantitive determination of nucleic acidsby using suitable calibrators. What is critical and limiting forclinical sensitivity (avoidance of false negative results) in this caseis the efficient purification of the nucleic acids (i.e. efficientbonding to the magnetic particle and the reversible release underPCR-compatible conditions).

A further object of the invention is a kit for performing a methodaccording to the invention that contains the following components:

-   -   (a) Reagents for dissolving the sample    -   (b) Magnetic particles containing silica or a suspension of        magnetic particles containing silica    -   (c) Washing buffer    -   (d) Elution buffer

The above lists and the following examples are applicable for theindividual components. Single or several components of the kit can alsobe used in a modified form.

With this invention it is possible by using specially producedsilica-coated magnetic particles to detect nucleic acids particularlyefficiently, automatically and quantitively from biological body samplepurifications using appropriate amplification techniques.

This invention thus represents an important contribution to nucleic aciddiagnostics.

EXAMPLES

The following are examples of protocols for performing the describedinvention. Exact reaction conditions for the respective nucleic acids tobe purified are given in these examples, but nevertheless variousparameters such as magnetic particle quantity, incubation temperatureand washing temperature, incubation and washing times and theconcentration of lysis buffer, washing buffer and elution buffer canvary depending on the particular nucleic acid to be purified.

Example 1 Production of Silicate-Coated Magnetite Particles fromBayoxide E 8706 Using Sodium Silicate 37/40 by the Gradual Reduction ofthe pH Value (Similar to the Method in WO 03/058649 A1)

Reaction Part:

4000 g of sodium silicate solution 37/40 (Cognis GmbH) is placed in a 6l three-neck flask with a KPG stirrer. 2000 g of Bayoxide 8706 (BayerAG) is added within ten minutes whilst stirring. Stirring then continuesfor one hour at room temperature.

Purification:

After the stirrer is switched off, the silica-coated magnetite beadssettle. This process can be accelerated if necessary by applying amagnetic field. After a waiting time of one hour, the supernatant isdrawn off. For purification, 4 l of water is added whilst stirring forapproximately ten minutes. The supernatant is again drawn off. Thiswashing process is repeated at least four times until the last washwater has achieved a pH value of 7.5-7.0.

Properties of the Silica-Magnetic Particles:

Zeta potential: −50.2 Silica content according to ESCA 7.0 atom % Si

Purity: The supernatant was colored yellow/brown after standing ten daysat room temperature.

Example 2 Production of Super-Pure Silica-Coated Magnetite Particlesfrom Bayoxide E 8706 Using Sodium Silicate 37/40 with a ContinuousReduction of the pH Value by Cross Flow Microfiltration

The reaction part described in Example 1 was repeated but the processingtook place not gradually or batchwise but instead with the aid of the“Centramate®” micro filtration unit from PALL with a 0.2 μm Supor®membrane cassette.

For this purpose, the magnetic particle suspension was drawn off via ahose by means of a pump and passed through the membrane cassette, withthe permeate being rejected but the retentate being fed back into thereaction vessel. The amount equivalent to the permeate was thenresupplied to the particle suspension.

After a filtration time of 12 h, the pH and conductivity of the permeatehad achieved the quality of the original water and the cleaning processwas ended.

Properties of the End Product:

Zeta potential: −41 mV Si content: 4.9 atom % Si determined according toESCA Silica content of the starting product Bayoxide 8706: 2.4 atom % Si

The differential amount, 2.5 atom % Si, was accordingly deposited on thesurface of the particles by silica treatment using sodium silicate. Thisproduces a silica layer thickness of 0.4 nm.

Purity: The particle suspension purified by ultrafiltration showed nodiscoloration in the supernatant even after standing for several monthsat room temperature.

Example 3 Production of Super-Pure Silicate-Coated Magnetite Particlesfrom Bayoxide E 8706 and Sodium Silicate 37/40 with a ContinuousReduction of the pH Value by Cross Flow Microfiltration Followed byFractionated Centrifugation

The end product described in Example 2 was centrifuged for seven minutesat 3225g with the aid of a centrifuge (Eppendorf 5810). Whereas the mainpart (>98%) of the product was sedimented, a dark brown coloredsupernatant remained that was discarded.

The residue was again added to water, centrifuged and separated from thecolored supernatant. This fractionated centrifugation was repeated eighttimes until the supernatant became colorless.

Properties of the End Product (Ninth Centrifugate):

Zeta potential: −35 mV Si content: 3.0% Si.

The differential amount, 0.6 atom %, was accordingly deposited on theparticle surface by silica treatment with sodium silicate. This resultedin a silicon layer thickness of 0.2 nm.

Purity: The supernatant of the magnetic particle suspension produced inthis way remained completely colorless even after storing for severalmonths.

This product quality showed outstanding values particularly with regardto magnetic separation. Thus, after applying a magnet an absolutelyclear supernatant was observed after less than twenty seconds.

Example 4 Optical Measurement of Aqueous Supernatants from Silica-CoatedMagnetic Particle Suspensions

In this experiment, the absorption spectra of two aqueous supernatantsof the silica-coated magnetic particles with lot designation HIE13266(originating from the inventive method of Example 2) and 3) and lotdesignation HIE12106R2 (originating from the method from WO 03/058649A1, based on Bayoxide E 8707) were recorded in a range of 221-750 nmusing a spectrometer from the Nanodrop company (see FIG. 1).

A water spectrum drawn from these spectra was used as a reference. Awater spectrum was again taken as a sample for control purposes (zeroline).

From the spectra, it could be seen that the aqueous supernatants of thesilica-coated magnetic particles HIE13266 had an absorption behaviorsimilar to water. On the other hand, the absorption lines of thesupernatants of HIE12106R2 showed a clearly changed and elevatedabsorption behavior up to a range of approximately 500 nm.

From this it can be seen that the new inventive production method withcontinuous washing (particles HIE13266) in a microfiltration unit led toreduced contamination effects or the occurrence of iron compounds in thesupernatant compared to particles HIE12106R2 with sequential multiplewashing or gradual reduction of the pH value (see also WO 03/058649 A1).These contamination effects with particles HIE12106R2 manifestthemselves by visible discoloration of the supernatants over time andalso increased absorption behavior. Furthermore, these reducedcontamination effects in the supernatant from the method according tothe invention indicate a closed silica layer on the particles.

Example 5 Behavior of Aqueous Supernatants of Silica-Coated MagneticParticles Using RT-PCR

The aqueous supernatants of the two differently silica-coated magneticparticles (lot designations HIE13266 and HIE12106R2) were processedusing magnetization. Particle lot HIE13266 was produced using theinventive production method with continuous washing in a microfiltrationunit (see Examples 2 and 3). Particle lot HIE12106R2 was produced byrepeated sequential washing (see WO 03/058649 A1) based on Bayoxide E8707. Both supernatants were then subjected to a quantitive RT-PCRintervention:

The so-called quantitative RT (reverse transcription)-PCR interventionwas carried out on the MX 4000 from Stratagene. As part of this, 5 μl ofthe supernatants of both of the particle supernatants, and 5 μl of wateras a control, was added to 20 μl of Mastermix. This contains thefollowing components: 400 nM Primer A, 400 nM Primer B, 10 ng MCF-7 RNA(Ambion), Taqman Primer 200 nM, 1× Buffer A, 5 mM MgCl₂; 1.2 mM dNTPs, 8U RNaseInhibitor, 20 U MuLV Reverse Transcriptase, 1.25 U Taq Gold (allfrom Applied Biosystems). The PCR program was: 30 min at 45° C., 10 minat 95° C., 45 cycles of 15 seconds at 96° C., 60 seconds at 63° C. and30 sec at 72° C.

The preparations were placed in a 96-well microtiter plate (Stratagene),sealed and placed in the analysis device. On completion of the run andusing device software, an individual C₁ value (number of cycle at whichthe selected base value intersects the amplification curve) was assignedto each sample at a selected basic value (fluorescence intensity) in theexponential amplification range of the signal curves.

As can be seen from FIG. 2, the amplification curves with supernatantsof particles HIE13266 are comparable with the amplification curves withwater as a sample. On the other hand, a shift of the amplificationcurves of approximately 3 C₁ values with supernatants from HIE12106R2can be seen on the right-hand side, which indicates interference ornegative influence on the efficiency of the RT-PCR.

1. Silica-coated magnetic particles, characterized in that they have aclosed surface coating with silicate with a maximum layer thickness of 5nm.
 2. The silica-coated magnetic particles as claimed in claim 1 with amaximum layer thickness of silicate of 2 nm.
 3. The silica-coatedmagnetic particles as claimed in claim 1 with a maximum layer thicknessof silicate of 0.5 nm.
 4. The silica-coated magnetic particles asclaimed in claim 1, with the magnetic material being iron oxide ormagnetite.
 5. The silica-coated magnetic particles as claimed in claim4, characterized in that the grain size distribution is between 0.1 and1 μm.
 6. A method for the production of particles as claimed in claim 1,characterized in that the initial silicate deposition from sodiumsilicate or silica sol on the magnetic particles is triggered by thesurface properties of the iron particles and the surface is thensmoothed and sealed by slow, continuous dilution and reduction of the pHvalue to neutral pH values.
 7. The method for the purification ofnucleic acids from biological body samples using magnetic particles asclaimed in claim
 1. 8. The method as claimed in claim 7, characterizedin that the nucleic acids are RNA or DNA.
 9. The method as claimed inclaim 7, characterized in that it relates to RNA of HCV or HIV.
 10. Themethod as claimed in claim 7, characterized in that the RNA or DNA isfrom fixed body samples.